The efficacy of the antimicrobial peptides D4E1, VvAMP-1 and Snakin1 against the grapevine pathogen aster yellows phytoplasma

The efficacy of the antimicrobial peptides

D4E1, VvAMP-1 and Snakin1 against the

grapevine pathogen aster yellows

phytoplasma

by

Nicole Spinas

Thesis presented in partial fulfilment of the requirements for the degree Master

of Science in Genetics at Stellenbosch University.

Supervisor: Prof. Johan T. Burger Co-supervisor: Dr. Dirk Stephan

Department of Genetics Faculty of AgriSciences

ii

Declaration

By submitting this thesis, I declare that the entirety of the work contained therein is my own, original work, and that I have not previously in its entirety or in part submitted it for

obtaining any qualification.

N Spinas Date

Stellenbosch University 2013

iii

Abstract

Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression.

The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv „Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv „Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system.

In total, 86 V. vinifera cv „Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a „recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv „Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf

iv material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups.

This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv „Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv „Chardonnay‟ vine.

v

Opsomming

Fitoplasma-siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan die onlangse ontdekking van fitoplasmas in Suid-Afrikaanse wingerde baie nadelige gevolge vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat in amper alle organismes as deel van hulle nie-spesifieke verdedigingsstelsel tot uitdruk kom. Hierdie peptiede kan beskerming bied teen ʼn wye verskeidenheid van bakteriële en swampatogene in plante. As gevolg van die feit dat fitoplasmas geen selmembraan of selwand het nie, word AMPe oorweeg as middel om weerstand te verleen teen hierdie fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die wingerd-patogeen aster vergeling fitoplasma (AYp) deur middel van Agrobacterium-bemiddelde tydelike uitdrukkingsisteme, te ondersoek.

Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer vanuit aartappel en wingerd) is gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-virus 35S uitdrukkingsvektore met vier verskillende AMP-koderende volgordes is dus ontwikkel. As ʼn alternatiewe metode om die moontlike effek van Vv-AMP1 op AYp in planta te toets, is enting van die Vv-AMP1 transgeniese Vitis vinifera cv „Sultana‟ plantmateriaal gedoen. Om hierdie AMPe se doeltreffenheid in hierdie tydelike uitdrukkingsvektore te toets, is pogings aangewend om die ruimtelike verspreiding en patogeenkonsentrasie van AYp in V. vinifera cv „Chardonnay‟ te beskryf. Verder is transmissie-eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana met AYp dmv die insekvektor, Mgenia fuscovaria, te infekteer. Plantmateriaal is getoets vir AYp in ʼn PCR met universele inleiers soos beskyf deur Grundersen en Lee (1996). Vir kwantifisering van die AYp infeksie, is „n semi-kwantitatiewe qPCR protokol geoptimiseer, met behulp van die SYBR Groen-gebaseerde stelsel. In totaal is 86 Chardonnay plantjies getoets vir AYp infeksie – twee-, drie-, vier-, sewe- en elf weke na die blootstelling aan die in vitro kondisies. Geen AYp infeksie kon egter opgespoor word nie en die plante het „n “herstel-fenotipe” vertoon.

vi Om die verspreiding van AYp in die arms van ʼn geïnfekteerde Chardonnay plant te ondersoek, is blare en ooreenstemmende internode van vyf lote getoets met PCR. Daar kon afgelei word dat, laat in die seisoen, AYp hoofsaaklik in die internode gevind word. In slegs enkele gevalle is fitoplasma-infeksies in blaarmateriaal, waarvan die ooreenstemmende internode negatief getoets het, gevind. Aangesien die AYp-geïnfekteerde wingerdmateriaal nie in vitro gekweek kon word nie, kon die effek van VvAMP-1 transgeniese wingerd nie teen AYp getoets word nie. AYp infeksies van C. roseus en N. benthamiana plante deur transmissie eksperimente met ʼn insekvektor was suksesvol. Toetse met tydelike uitdrukkingsvektore is uitgevoer op die AYp-geïnfekteerde N. benthamiana materiaal. Kwantifisering van fitoplasma in hierdie materiaal het die afname van AYp in beide die AMP behandelingsgroep en die kontrole groep getoon.

Hierdie studie het ʼn qPCR-toets geoptimiseer om geïnfekteerde plantmateriaal met AYp op te spoor en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in hierdie studie gebruik is, het geen beduidende effek van die AMPe op AYp konsentrasie getoon nie. Hierdie studie het bewys dat AYp nie instand gehou kan word deur in vitro kweking van Chardonnay materiaal nie, en dat weefselkultuur dus ʼn manier kan wees om AYp in hierdie kultivar te elimineer. Sover ons kennis strek, is hierdie studie die eerste om die ruimtelike verspreiding van AYp in arms van geïnfekteerde wingerdstokke, te rapporteer.

vii

Abbreviations

kDa kilo Dalton

kPa kilo Pascal

kV kilo Volt fg femtogram µF microfarad µl microliter µM micromolar min minute ng nanogram Ω ohm sec second °C Degrees Celsius

DNA Deoxyribonucleic acid

GUS β-glucuronidase

IWBT Institute for Wine Biotechnology

KCl Potassium chloride

KH2PO4 Potassium di-hydrogen phosphate

MgCl2 Magnesium chloride

viii

MS Murashige and Skoog

NaCl Sodium chloride

Na2HPO4 Disodium hydrogen phosphate

NaH2PO4 Monosodium phosphate

Na2EDTA Diaminetetraacetic acid

OD Optical density

PCR Polymerase chain reaction

rDNA Ribosomal deoxyribonucleic acid

SA South Africa

SDS Sodium dodecyl sulphate

SN1 Snakin1

Tris-HCL Tris-hydrochloride

UV Ultra-violet

qPCR quantitative real-time PCR

V Volts

Vv-AMP1 Vitis vinifera-antimicrobial peptide 1

W Watt

ix

Acknowledgements

I would like to express my sincerest gratitude and appreciation to the following people and institutes:

 My supervisor Prof. Johan T. Burger for giving me the opportunity to perform this research and his continuous guidance and support throughout the study.

 Dr. Dirk Stephan for his intellectual input and guidance.

 My colleagues and friends in the Vitis laboratory for their input and their encouragement and help through tough times.

 The staff at the IWBT for allowing me to use their facilities and for friendly assistance.

 Winetech and DAAD for financial assistance.

 My family, for their continuous support, love and encouragement throughout this study.

 My mother, for never giving up on me, for reading though endless hours of my thesis, for her continuous support, love and encouragement throughout this study.

 Rudy, for endless patients, encouragement and support.  My heavenly Father

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Table of Contents

1.1 Background and motivation for this study………..……….………...1

1.2 Project proposal………..……….………...2

1.3 References………..……….………...2

Chapter 2: Literature review……….………….…...….3

2.1 Introduction……….……….…………3

2.2 Phytoplasmas………...………..….…………..…3

2.2.1 The discovery of phytoplasmas…...………..………....3

2.2.2 Classification of phytoplasmas...………...…………...4

2.2.3 Plant hosts………..…….…..…6

2.2.4 Dual life cycle………..………...6

2.2.5 Insect vector...7

2.2.6 Symptoms………...…...…....7

2.2.7 Interactions of phytoplasmas with their hosts...…...………...…8

2.2.8 Detection methods………...…...9

2.2.8.1 Past- Biological diagnostic approaches...10

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2.2.9 Seasonal and spatial distribution……….…11

2.2.10 Control strategies………...12

2.2.10.1 Auxin-induced recovery...12

2.2.10.2 Natural recovery...12

2.2.10.3 Hot water treatment...13

2.2.10.4 Abiotic stresses...13

2.2.10.5 Expression of antimicrobial peptides transgenic in plants...14

2.3 Antimicrobial peptides ……….….14

2.3.1 General Information……….……….…………...15

2.3.2 Plant AMPs……….……….………...15

2.3.3 Mechanism of cell death induced by AMPs...16

2.3.4 Exploiting AMPs in plant disease control...18

2.3.4.1 Transgenic plants expressing AMPs...18

2.3.4.2 Transient expression of AMPs...19

2.3.5 Factors influencing AMP expression...20

2.4 Conclusion……….……….……….…...…....20

2.5 References……….….21

Chapter 3: Establishing aster yellows phytoplasma infected plant material………...36

3.1 Introduction……….……….………..…36

3.2 Materials and methods……….………..……39

3.2.1 Vitis vinifera plant material……….……….……...39

3.2.2 Nicotiana benthamiana plant material……….……...…40

3.2.3 Catharanthus roseus plant material……….…...41

3.2.4 Diagnostic nested-PCR used to detect AYp………..…...41

3.3 Results……….……….……….……...42

3.3.1 Establishment of AYp-infected V. vinifera material…………..……...42

3.3.2 Establishment of AYp-infected N. bethamiana material………..…...44

3.3.3 Establishment of AYp-infected C. roseus material………..…...…44

3.4 Discussion……….……….……….…...45

3.5 References……….……….……….…...…48

Chapter 4: Spatial distribution of AYp in V. vinifera cv. Chardonnay………...…….54

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4.2 Materials and methods……….……….…………....….56

4.2.1 Plant material……….……….…………....….56

4.2.2 Diagnostic PCR……….……….……….56

4.2.3 Quantitative analysis……….………..57

4.3 Results……….……….……….……...58

4.3.1 Spatial distribution of AYp……….………...58

4.3.2 Quantification of AYp……….……….………...60

4.4 Discussion……….……….………...…...63

4.5 References……….……….……….………...65

Chapter 5: Antimicrobial peptides and their in planta activity against AYp………...68

5.1 Introduction……….………..………...68

5.2 Materials and methods……….………….………….…………...70

5.2.1 Candidate antimicrobial peptides……….……….…………...…...70

5.2.2 Isolation of Snakin1……….………...71

5.2.2.1 Snakin1 isolation from potato tubers………...71

5.2.2.2 Snakin1 isolation from Chardonnay…….………...72

5.2.3 AMP expression vector constructs……….………...73

5.2.3.1 Vv-AMP1 and D4E1 expression vectors...………....…73

5.2.3.2 SN1-Chardonnay and SN1-Potato expression constructs...74

5.2.4 Transformation of Agrobacterium cells……….………...74

5.2.5 Agro-infiltration of plants……….………...74

5.2.5.1 Agro-infiltration of 35S:GUSi...74

5.2.5.2 Agro-infiltration of the AMPs...75

5.2.6 GUS assay……….……….………....….76

5.2.7 Screening the in planta activity of AMPs against AYp in N. Benthamiana……….………...76

5.2.8 Peptide expression……….……….……....….77

5.2.8.1 Protein extractions……….………...78

5.2.8.2 Western blot……….………...78

5.2.9 The effect of Vv-AMP1 on AYp through in vitro grafting………....….79

5.3 Results………...….79

5.3.1 Snakin1 isolation from potato and grapevine...79

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5.3.3 GUS expression in V. vinifera and C. roseus………...…..80

5.3.4 In planta activity of AMPs against AYp………...……...82

5.3.5 Peptide expression…..……….……….……...84

5.3.5 The effect of Vv-AMP1 on AYp through in vitro grafting……..…...87

5.4 Discussion……….……….………....88

5.5 References……….……….…….………...91

Chapter 6: General conclusion……….………….………….…………...……96

xiv

List of Figures

Figure 1: The dual life cycle of phytoplasmas (Christensen et al., 2005)...pg6

Figure 2: Phytoplasma-associated symptoms in grapevine. (A) A grapevine branch

displaying yellowing of the leaves. At the end of the branch, bunch abortion of growth tip can also be observed (Photo taken by J Joubert from VinPro, South Africa). (B) Grapevine showing aborted fruits as well as yellowing and necrosis in leaf veins (Photo taken by Dr RE Davis of the Molecular Plant Pathology Laboratory, Unites States Department of Agriculture)...pg8

Figure 3: The processes of pore formations by AMPs. A: The barrel-steve mechanism. B:

The carpet mechanism (Pelegrini et al., 2011)...pg17

Figure 4: Modes for antimicrobial peptide activity. (Gallo and Huttner, 1998)...pg18

Figure 5: Tissue culture V. vinifera cv „Chardonnay‟ plants cultured in MS media. Plantlets

were kept at a 16h light and 8h dark photoperiod at 23°C and 19°C...pg39

Figure 6: Phloem scrapings and leaf material collected from in vitro V. vinifera cv

„Chardonnay‟ plants. Phloem scrapings were taken using a scalpel blade... pg40

Figure 7: A) Insect vector M. fuscovaria. B) N. benthamiana containing five insects kept

in a cage for two days. C) N. benthamiana plant 4 weeks after the transmission experiment...pg41

Figure 8: Contamination rate seen in Chardonnay plants once placed in vitro, collected from

the vineyard during five different time intervals. Plants collected in January showed a 59% contamination rate after 2 month. This contamination rate increased to 63% and 70% when plats were collected in February. From March onwards, all plants placed in vitro developed 100% contamination after 2 weeks ...pg43

xv

Figure 9: Agarose gel-electrophoresis of nested-PCR products. Lane 1: 1kb Molecular

marker. Lane 2: Positive control. Leaf material collected from a V. vinifera cane before being placed in vitro. Lane 3, 5, 7, 9: Phloem scrapings from in vitro material. Lane 4, 6, 8, 10: Leaf material from in vitro material. Lane 11: Healthy V. vinifera leaf material. Lane 12: No-template control...pg43

Figure 10: Agarose gel electrophoresis of nested-PCR products. Lane 0 and last lane: 1kb

Molecular marker. Lane 1-3: Healthy N. benthamiana. Lane 4, 6 and 9: Healthy N. benthamiana after transmission experiment. Lane 5, 7 and 9: AYp -infected N. benthamiana after transmission experiment. +: Positive control. -: Negative control. NTC: no-template control after the first PCR and nested-PCR...pg44

Figure 11: A) Aster yellows phytoplasma-infected C. roseus plant infected through natural

transmission of the insect vector M. fuscovaria. B) Healthy C. roseus plant grown in the greenhouse...pg45

Figure 12: Leaf material and respective phloem scrapings taken from all five canes and

stored at -80°C...pg56

Figure 13: Spatial distribution of AYp in five canes (A-E) of the same V. vinifera cv

„Chardonnay‟ plant from Vredendal, South Africa. Node and leaf samples were tested on each cane for the presence of AYp. Samples labelled (A, B, B1, D, D1, D2, E, E1, E2) were run on a PCR as an internal control for the 18S rDNA of V. vinifera. These results are discussed below...pg59

Figure 14: Agarose gel electrophoresis of PCR products. Lane 0: 1kb molecular marker.

Lane 1-18: 18 samples of V. vinifera cv „Chardonnay‟ material circled in blue from Figure 2. The subscripts L and N stand for leaf and node material. + : Positive control. NTC: no-template control...pg60

xvi

Figure 15: Amplification profile of the dilution series. 1ng 0.1ng 0.01ng 1X10 -3

ng 1X10-4ng 1X10-5ng 1X10-6ng...pg61

Figure 16: The standard curve resulting from the CT values of each triplicate plotted against

the concentrations of each sample...pg61

Figure 17: Amplification curve of AYp-infected V. vinifera plant material collected from the

same vine in different seasons. pAY61. V. vinifera collected in October 2011. AYp-infected V. vinifera collected in April 2012. Healthy V. vinifera. No-template control...pg62

Figure 18: Melt curve of AYp-infected V. vinifera plant material collected from the same

vine in different seasons. pAY61. V. vinifera collected in October 2011. AYp-infected V. vinifera collected in April 2012. Healthy V. vinifera. No-template control...pg63

Figure 19: Modes for antimicrobial peptide activity (Gallo and Huttner, 1998). A:

AMPs may form pores through which ions leak out, causing the energy gradients to dissipate and leading to cell lysis (Bowman et al., 2003). B: AMPs bind to intracellular targets within the bacterial cell which causes a decrease in protein synthesis, leading to cell death (Park et al., 1998)...pg68

Figure 20: Agro-infiltration on N. benthamiana using an AMP expression vector and a

control. The AMP expression vector was infiltrated on one side of the main vein and the control vector on the opposite side...pg76

Figure 21: Grafting procedure performed under the microscope, using a scalpel blade and

xvii

Figure 22: The binary vector pCB301. oriV: origin of replication. nptIII: neomycin

phosphotranferase gene. trfA: part of the origin of replication. RB: right border. MCS: multiple cloning site. LB: left border. CaMV 35S: 35S promoter from cauliflower mosaic virus. Snakin1: Snakin1-Chardonnay or Snakin1-Potato. 35S Term: 35S termination signal. The AMP-containing 35S expression cassette (outlined in red) was cloned into the pCB301 binary vector using the restriction enzymes SacI and PstI (indicated in the MCS of pCB301)...pg80

Figure 23: GUS expression observed in V. vinifera cvs „Chardonnay‟ and „Chenin blanc‟ leaf

material. A: V. vinifera cv „Chenin blanc‟ leaves infiltrated with 35:GUSi 6dpi (top) and the negative control (bottom). B: V. vinifera cv „Chardonnay‟ leaf material leaves infiltrated with 35:GUSi 6dpi (top) and the negative control (bottom)...pg81

Figure 24: GUS expression observed in C. roseus leaf material. A: C. roseus leaves

infiltrated with 35:GUSi 6dpi at 30kPa for 15min. B: C. roseus leaves infiltrated with 35:GUSi 6dpi at 50kPa for 10min.C: C. roseus leaves infiltrated with 35:GUSi 6dpi at 90kPa for 2min. D: Negative control...pg82

Figure 25: SDS-PAGE stained with Coomassie blue. Lanes 1+10: Low weight molecular

marker. Lanes 2-4: N. benthamiana leaf material infiltrated with Vv-AMP1. Lanes 5+6: N. benthamiana leaf material infiltrated with SN1-Chardonnay. Lanes 7+8: N. benthamiana leaf material infiltrated with SN1-Potato. Lane 9: N. benthamiana leaf material infiltrated with the control construct pBin61S...pg85

Figure 26: Western blot results for Vv-AMP1, SN1-Chardonnay and SN1-Potato expression

in N. benthamiana plants. (A) M: Low weight molecular marker. Lane 1: Vv-AMP1 expression in infiltrated N. benthamiana leaves. Lane 2: Control infiltration using pBin61S. (B) M: Low weight molecular marker. Lane 1: SN1-Chardonnay expression in infiltrated N. benthamiana leaves. Lane 2: SN1-Potato expression in infiltrated N. benthamiana leaves. Lane 3: Control infiltration using pBin61S. The four antibodies designed to recognize the antigen region KDKKNSKGQP, displayed these results. (C): M: Low weight molecular marker. Lane 1: SN1-Chardonnay expression in infiltrated N. benthamiana leaves. Lane 2: SN1-Potato expression in infiltrated N. benthamiana leaves. Lane 3: Control infiltration using pBin61S. The two antibodies

xviii designed to recognize the antigen regions PSGTYGNKHE and EECKCVPSGT, displayed these results......pg86

Figure 27: Chenin blanc and Chardonnay shoots grafted onto sterile Vv-AMP1 transgenic

Sultana. A: Healthy Chenin blanc grafted onto Vv-AMP1 transgenic Sultana. B: Healthy Chardonnay grafted onto Vv-AMP1 transgenic Sultana...pg87

xix

List of Tables

Table 1: 16S rRNA group-subgroup classification and „Candidatus Phytoplasma‟ species (Dr

RE Davis, Unites States Department of Agriculture, Phytoplasma Resource Centre) ...pg5

Table 2: Primers used for the detection of the 18S rDNA of V. vinifera plants and for the

quantitative real-time PCR analysis to determine AYp titre...pg57

Table 3: Number of leaf and node samples collected from all five vines from one

AYp-infected V. vinifera cv „Chardonnay‟ plant...pg58

Table 4: AYp infection detected in leaf and node material in five canes from one

AYp-infected V. vinifera cv „Chardonnay‟ plant...pg59

Table 5: Mean threshold cycles (CT) of standard pAY61 seen in all seven dilutions run in

triplicate. The genome unit (GU) for each dilution are also shown together with the CT

standard deviation calculated for each sample run in triplicate...pg61

Table 6: Primers used to amplify Snakin1 from potato. Restriction enzyme recognition

sequences (underlined) and translation enhancer sequence (bold) are

indicated....pg71

Table 7: Primers used to amplify Snakin1 from V. vinifera cv „Chardonnay‟. Restriction

enzyme recognition sequences (underlined) and translational enhancer sequence (bold) are identified...pg72

Table 8: List of primers used during Vv-AMP1 and D4E1 expression vector construction.

The restriction enzyme recognition sites (underlined) and translational enhancer sequences (bold) are indicated...pg73

xx

Table 9: The original SN1-Chardonnay amino acid sequence sent to Abmart for antibody

production. The potential antigenic regions used by Abmart (China) to design six primary antibodies for the detection of Snakin 1 are listed below...pg77

Table 10: Antimicrobial peptides used for the transient expression in an AYp-infected N.

benthamiana plant. AY titres were detected and analysed by quantitative PCR...pg83

Table 11: CT values obtained from qPCR profiles of N. benthamiana plants infected with

AYp that were treated with Vv-AMP1, D4E1, SN1-Chardonnay and SN1-Potato and the untreated control plants. The statistical differences between the two treatment groups are shown by the p-value. CT values obtained from AMP infiltrated leaf areas

1

Introduction

1.1 Background and motivation for this study

The importance of grapevine as an agricultural commodity in SA cannot be over emphasized. More than 115 000 hectares of land in SA are planted to grapevine and the South African wine industry contributed 417.5 million gross litres of wine for sale to private and producer cellars in 2011, with an increase of 23.9% estimated for 2012 (http://www.sawis.co.za). Phytoplasma diseases are known to have caused disastrous effects in vineyards in European countries, resulting in significant reductions in fruit yield and wine quality (Lee et al., 2000). Therefore, the recent discovery of phytoplasma infections in SA could be highly problematic to the South African wine industry. It is therefore of high importance to find an approach to control this disease. A long term approach to control this pathogen through the development of resistance is desirable and should be investigated and implemented. The current study intends to explore an approach to induce resistance against the grapevine pathogen aster yellows phytoplasma (AYp), to control this devastating new disease.

Scientists have started employing short peptides, known as antimicrobial peptides (AMPs), to combat plant pathogens. These small molecules of less than 50 amino acids in length are expressed by almost all organisms as part of their non-specific defence system (Montesinos, 2007). Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time-consuming and therefore the pre-screening of potential AMPs is necessary. In vitro pre-screening of AMP activity is valuable, but is impossible for phytoplasmas since these pathogens cannot be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against phytoplasma pathogens.

In this study, a transient expression system described by Visser et al. (2012) was used to test the in planta activity of four AMPs against the grapevine pathogen AYp. This system can be used as an in planta pre-selection for AMP efficacy and can be performed in a relatively short time period, for a large number of AMPs. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in Vitis vinifera cv „Chardonnay‟ material.

2

1.2 Project proposal

This study aimed to test the in planta activity of AMPs against the grapevine pathogen AYp through a transient expression system.

To achieve the proposed aim, it was necessary to reach the following objectives:

 Test the expression of foreign genes in grapevine using Agrobacterium-mediated transient expression vectors containing the GUS control gene

 Construct Agrobacterium-mediated transient expression vectors containing AMP genes and test for the expression of these genes

 Identify and establish in vitro phytoplasma-infected plants

 Conduct transmission experiments using the vector Mgenia fuscovaria on Nicotiana benthamiana and Catharanthus roseus

 Infiltrate phytoplasma-infected plants with the AMP expression constructs  Graft phytoplasma-infected plants onto existing Vv-AMP1 transgenic plants  Test the effects of the AMPs by measuring microbial titres and disease development

 Determine the distribution of AYp in the canes of an infected grapevine plant

1.3 References

Lee IM, Davis RE, Gundersen-Rindal DE (2000) Phytoplasma: phytopathogenic mollicutes. Annual Review of Microbiology 54: 221-255

Montesinos E (2007) Antimicrobial peptides and plant disease control. FEMS Microbiology Letter 270 (1): 1-11

Visser M, Stephan D, Jaynes JM, Burger JT (2012) A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens. Letters in Applied Microbiology 54 (6): 543-551

Internet resources

3

Chapter 2

Literature review

2.1 Introduction

About 1 000 years ago the Chinese were great admirers of the obscure bacteria phytoplasma. They found the symptoms in peonies so attractive, that the Song Dynasty‟s imperial court was given a special annual tribute consisting of these infected flowers (Strauss, 2009). Most of the effects displayed by these microbes, are however far from pretty. In the European countries alone, phytoplasma infections have caused devastating yield losses in several fruit crops. During only one phytoplasma outbreak in apple trees in 2001, Germany lost €25 million, while Italy made a loss of €100 million (Strauss, 2009). This bacterium is however not only causing effects in the European countries. In Africa and the Caribbean, infected palm trees are causing people to have insufficient nourishment and building materials (Maramorosch, 2011; Strauss, 2009). In grapevine, phytoplasmas are known to have caused disastrous effects in vineyards in European countries, resulting in significant reductions in fruit yield and wine quality (Lee et al., 2000). In 2006, Botti and Bertaccini discovered the first ever mixed phytoplasma infection in South African vineyards. The South African wine industry contributed R2.6 billion to the country‟s gross domestic product in 2008 and employs over 275 000 people (http://www.sawis.co.za). Due to the importance of the grapevine industry on the South African economy, it is crucial to combat all pathogens including the recently discovered phytoplasma. This chapter will give some background information on phytoplasmas and antimicrobial peptides, which are molecules used for inducing pathogen resistance in plants.

2.2 Phytoplasmas

2.2.1 The discovery of phytoplasmas

In 1926, Kunkel described a disease that destroys crops, orchards and ornamental plants. For several reasons, scientists believed the disease was caused by a virus or viruses, as the pathogen could not be cultured in vitro, was transmitted by insects and displayed symptoms similar to a virus infection (Doi et al., 1967). For the next 40 years scientists examined the disease but were unsuccessful in finding a virus.

4 When Maramorosch (1958) injected insects with the antibiotic tetracycline and the infectious agent phytoplasma, the injected insects did not transmit aster yellows to the plants. Knowing that antibiotics had no effect on viruses, he concluded that the high temperatures in the greenhouse, rather than the drug prevented pathogen transmission. In 1967, Doi and colleagues discovered structures resembling those of mycoplasmas and termed the causal agent mycoplasma-like organisms (MLOs). Mycoplasmas are small groups of typically parasitic bacteria that lack cell walls and can cause diseases in plants, humans and animals. In 1994, this mycoplasma-like organism was given the name phytoplasma by the Phytoplasma Working Team at the 10th Congress of the International Organization of Mycoplasmology.

2.2.2 Classification of phytoplasmas

Phytoplasmas diverged from gram-positive ancestors and belong to the class Mollicutes. They are petite, cell wall-less pleiomorphic bacteria of approximately 500nm in diameter (Lee et al., 1998). Even though phytoplasmas have a smaller genome compared to most bacteria, they manage a very complex life cycle that involves two noticeably different environments – plants and insects. Early diagnostic approaches distinguished phytoplasma infections from other grapevine diseases, by observing the main symptoms that phytoplasma diseases express in plants (Gasparich, 2009). As symptom expression is quite uniform among different phytoplasma species however, symptomatology cannot be used to distinguish one phytoplasma species from another. Focus has therefore shifted to a molecular approach of grouping this pathogen. Phytoplasmas are currently being classified and grouped into different subgroups according to the sequence of their 16S ribosomal RNA (rRNA) genes (Seemüller et al., 1998). The table shown below classifies phytoplasmas into „Candidatus Phytoplasma‟ species based on the nucleotide sequence of the 16S rRNA gene. Each 16S rRNA group represents at least one distinct „Candidatus Phytoplasma‟ species (Table 1). These main groups of phytoplasma species can further be classified into sub-groups, which share ≥97% similarity in their 16S rRNA sequences. Strains found in a specific group are known to have substantial genetic variations and occupy diverse ecological niches (Gundersen et al., 1996; Seemüller et al., 1994; Seemüller et al., 1998).

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Table 1: 16S rRNA group-subgroup classification and „Candidatus Phytoplasma‟ species (Dr RE Davis, Unites States

Department of Agriculture, Phytoplasma Resource Centre)

Phytoplasma/disease common name 16S rRNA group-subgroup GenBank no. Named 'Candidatus Phytoplasma' species Informally proposed 'Candidatus Phytoplasma' species

Aster yellows (AY) 16SrI M30790 'Candidatus Phytoplasma asteris'

WB disease of lime 16SrII-B U15442 'Ca. Phytoplasma aurantifolia'

Western X-disease 16SrIII-A L04682 'Ca. Phytoplasma pruni'

Palm lethal yellowing 16SrIV-A U18747 'Ca. Phytoplasma palmae'

Elm yellows 16SrV-A AY197655 'Ca. Phytoplasma ulmi'

Jujube WB 16SrV-B AB052876 'Ca. Phytoplasma ziziphi'

Flavescence dor�e 16SrV-C AF176319 'Ca. Phytoplasma vitis'

Clover proliferation 16SrVI-A AY390261 'Ca. Phytoplasma trifolii'

Ash yellows 16SrVII-A AF092209 'Ca. Phytoplasma fraxini'

Loofah WB 16SrVIII-A AF086621 'Ca. Phytoplasma luffae'

Almond lethal disease 16SrIX-D AF515636 'Ca. Phytoplasma phoenicium'

Apple proliferation 16SrX-A AJ542541 'Ca. Phytoplasma mali'

Pear decline 16SrX-C AJ542543 'Ca. Phytoplasma pyri'

Spartium WB 16SrX-D X92869 'Ca. Phytoplasma spartii'

European stone fruit Y 16SrX-F AJ542544 'Ca. Phytoplasma prunorum'

Rice yellow dwarf 16SrXI-A AB052873 'Ca. Phytoplasma oryzae'

Stolbur phytoplasma 16SrXII-A AF248959 'Ca. Phytoplasma solani'

Australian GY 16SrXII-B Y10097 'Ca. Phytoplasma australiense'

Hydrangea phyllody 16SrXII-D AB010425 'Ca. Phytoplasma japonicum'

Strawberry yellows 16SrXII-E DQ086423 'Ca. Phytoplasma fragariae'

Mexican periwinkle Vir 16SrXIII-A AF248960 No 'Candidatus' name proposed

Bermuda grass WL 16SrXIV AJ550984 'Ca. Phytoplasma cynodontis'

Hibiscus WB 16SrXV AF147708 'Ca. Phytoplasma brasiliense'

Sugarcane yellow leaf 16SrXVI AY725228 'Ca. Phytoplasma graminis'

Papaya bunchy top 16SrXVII AY725234 'Ca. Phytoplasma caricae'

Potato purple top wilt 16SrXVIII DQ174122 'Ca. Phytoplasma americanum'

Chestnut WB 16SrXIX AB054986 'Ca. Phytoplasma castaneae'

Buckthorn WB 16SrXX X76431 'Ca. Phytoplasma rhamni'

Pine shoot proliferation 16Sr XXI AJ632155 'Ca. Phytoplasma pini'

Nigerian Awka disease 16Sr XXII-A Y14175 'Ca. Phytoplasma cocosnigeriae‟

Buckland Valley GY 16SrXXIII-A AY083605 No 'Candidatus' name proposed

Sorghum bunchy shoot 16SrXXIV-A AF509322 No 'Candidatus' name proposed

Weeping tea WB 16SrXXV-A AF521672 No 'Candidatus' name proposed

Sugarcane yellows phytoplasma D3T2

16SrXXVII-A AJ539180 No 'Candidatus' name proposed

Derbid phytoplasma 16SrXXVIII-A AY744945 No 'Candidatus' name proposed

Cassia italica WB 16SrXXIX EF666051 'Ca. Phytoplasma omanense'

Salt cedar WB 16SrXXX FJ432664 'Ca. Phytoplasma tamaricis'

Parsley leaf of tomato " EF199549 'Ca. Phytoplasma lycopersici'

Tanzanian lethal disease " X80117 'Ca. Phytoplasma cocostanzaniae'

Chinaberry yellows " AF495882 No 'Candidatus' name proposed

 Abbreviations are as follows: AY, aster yellows; WB, witches'-broom; Y, yellows; GY, grapevine yellows; Vir, virescence; WL, white leaf.

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2.2.3 Plant hosts

To date, phytoplasmas have been found to infect several dicotyledonous-, cultivated- and wild plant species worldwide (Hollingsworth et al., 2008). Apple, celery, china asters, grapevine, carrots, lettuce, periwinkle, potato and redcurrant are just some examples of plant hosts that phytoplasmas are known to infect (Kuske and Kirckpatrick, 1992; Schneider et al., 1993; Tanne and Orenstein, 1997; Orenstein et al., 1999; Lee et al., 1993; Seemüller et al., 1994; Přibylová et al., 2011). Different phytoplasma species have been shown to infect Vitis vinifera including flavescence dorée (FD), bois noir (BN), Australian grapevine yellows phytoplasma (AGYp) and aster yellows phytoplasma (AYp). In South Africa, the phytoplasma strain causing yellows disease in infected vines was found to be AYp (Engelbrecht et al., 2010). AYp is known to infect over 300 plant species from 48 different plant families around the world (Stansbury et al., 2001). To date, AYp infections have been observed in vineyards in the Waboomsrivier area near Rawsonville and in the Olifants River area in the Vredendal district of SA.

2.2.4 Dual life cycle

Phytoplasmas can replicate in two distinctively different hosts - plants and insects (Figure 1). In plants they reside in the cytoplasm of sieve cells of the phloem, and in their insect vectors they are found in various organs inside and outside of the cells (Doi et al., 1967).

Figure 1: The dual life cycle of phytoplasmas (Christensen et al., 2005).

During the latent period, the insect vector acquires the pathogen from the plant host. It then takes ~ 3 weeks till the phytoplasma titres reach the infectious level.

7 During the inoculation feeding, the infectious insect introduces the phytoplasma into a healthy plant. This process can take between 7 and 80 days (Murral et al., 1996). Phytoplasmas are transferred with saliva into the punctured sieve element of the plant. From here the pathogen then spreads systematically throughout the plant, using the continuous sieve tube system. As phytoplasmas replicate in both plants and insects and cannot be cultured in vitro, they are very challenging pathogens to study.

2.2.5 Insect vector

Insect vectors for phytoplasma transmission include the leafhopper and plant hopper families. In 2011, Krüger and colleagues discovered that the vector for AYp transmission in grapevine in SA was the insect Mgenia fuscovaria. Studies have shown that phytoplasma strains in insect vectors and plants vary greatly. The plant host range depends less on the phytoplasma strain, and more on the natural insect vector species that are capable of transmitting the phytoplasma, and by the feeding behaviour of the insect vectors (McCoy et al., 1989; Kunkel, 1926; Grylls, 1979). Phytoplasmas can have a low insect vector specificity or high insect vector specificity, meaning that they can be transmitted by one or more insect vectors at a given time (Christensen et al., 2005). It is also known that insect vectors can transmit more than one type of phytoplasma and that plants can be infected by two or more distinct phytoplasmas at the same time. The geographic distribution of various insect vectors and preferred plant hosts of each vector, are the two major factors that determine whether a specific plant will be infected by one, or by multiple phytoplasmas (Lee et al., 1998).

2.2.6 Symptoms

Grapevine plants show basically the same type of symptoms, regardless of the infecting phytoplasma species (Belli et al., 2010). Some cultivars of grapevine may be more or less tolerant and may therefore show milder symptoms or no symptoms at all. The grapevine cultivar „Chardonnay‟ is highly susceptible to several different phytoplasma infections, and is thus very useful in the successful identification of affected plants in the vineyard (Gibb et al., 1999; Orenstein et al., 2001). In grapevine, symptoms of phytoplasma infections can be observed in the leaves, canes and bunches (Figure 2).

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Figure 2: Phytoplasma-associated symptoms in grapevine. (A) A grapevine branch displaying yellowing of the

leaves. At the end of the branch, bunch abortion of growth tip can also be observed (Photo taken by J Joubert from VinPro, South Africa). (B) Grapevine showing aborted fruits as well as yellowing and necrosis in leaf veins (Photo taken by Dr RE Davis of the Molecular Plant Pathology Laboratory, Unites States Department of Agriculture).

In early spring, vines may show irregular sprouting and then at the onset of summer, leaves start to roll downwards and become yellow in white-berried cultivars, and purple-reddish in red-berried cultivars (Belli et al., 2010; Gibb et al., 1999; Mitrev et al., 2007; Orenstein et al., 2001; Stansbury et al., 2001; Strauss, 2009). The berries then start to wither and the bunches dry up, while the canes develop irregularly or not at all (Belli et al., 2010; Radonjić et al., 2009; Magarey and Wachtel, 1982). Symptoms of phytoplasma infections can be limited to a sector or a branch, whereas the remaining plant looks normal. Phytoplasmas also promote vegetative growth and dwarfism, but hinder reproductive activities in the infected plant (Strauss, 2009). „Witches broom‟ and phyllody is another symptom seen in phytoplasma-infected sink tissues (Bertaccini, 2007; Hogenhout and Loria, 2008). Two or more phytoplasma species can infect the same vine simultaneously. These mixed infections do however not show differences in symptomatology to a single infection, which makes visual evaluation most difficult. The exact interaction of the pathogen with the host plant is still unknown, but the symptoms suggest that phytoplasmas interfere with fundamental cellular and developmental pathways in plants (Hogenhout et al., 2008).

2.2.7 Interaction of phytoplasmas with their hosts

Phytoplasma infections impact the plant negatively; however it may or may not affect the fitness and survival of the insect vector (Hogenhout et al., 2008). Some

9 of the morphological changes seen in infected plants attract insect vectors, and certain insects live longer and have more progeny on AYp-infected plants (Hogenhout et al., 2008). This suggests that the pathogen doesn‟t only interfere with the plant‟s fundamental pathways, but also down-regulates the plant‟s defence against leafhoppers (Sugio et al., 2011). According to recent studies, phytoplasmas induce phenotypic changes in plants through the production of effector proteins (Bai et al., 2009). To-date, 56 secreted AY-witches‟ broom proteins, also called SAPs, have been identified that are candidate effector proteins. These proteins are secreted into the plants cytoplasm by the Sec-dependent protein translocation pathway, similar to Gram-positive bacteria. Once the proteins have been discharged into the phloem they target other plant cells by symplastic transport (MacLean et al., 2011). In 2008, Hogenhout and her colleagues discovered SAP11, a protein secreted by AY-witches‟ broom, which accumulated in the plant cell nuclei and alters plant cell gene activity. More recently, SAP11 has also been shown to destabilize class II CINCINNATA- related TCP transcription factors, resulting in the crinkled leaf and witches‟ broom phenotype (Sugio et al., 2011). Another effector protein discovered in onion yellows phytoplasma, namely TENGU, induces symptoms of witches‟ broom and dwarfism in plants, and is also thought to interfere with the plants auxin-related pathways, thereby affecting plant development (Hoshi et al., 2009). MacLean and co-workers (2011) discovered SAP45, which has been found to interfere with floral development, another symptom of AY-witches‟ broom. It is clear that phytoplasmas secrete effector proteins that function inside the hosts cells. The extent to which phytoplasmas rely on these proteins to influence their diverse plant and insect hosts still remains unclear. However, from research done, scientists have discovered new hope for unravelling the pathogenicity mechanism of phytoplasmas.

2.2.8 Detection methods

The importance of being able to reliably distinguish phytoplasmas from similar grapevine diseases, and for discriminating different phytoplasmas from one-another, has attracted the attention of researchers worldwide. This activity has led to the development of a series of detection techniques, which have evolved from biological diagnostic approaches to molecular protocols (Belli et al., 2010).

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2.2.8.1 Past – Biological diagnostic approaches

Based on phytoplasma symptoms, one can generally distinguish phytoplasma infections from other grapevine disorders, for example leafroll disease (Belli et al., 2010). Symptom expression is however quite uniform amongst phytoplasma species and can thus not be used to reliably distinguish one phytoplasma species from another. Indexing techniques were therefore applied on the hybrid Baco 22A, but did not help much as the symptomatic response induced by different phytoplasmas in Baco 22A is similar (Belli et al., 2010). Successful transmission to Baco 22A was used to distinguish between FD and BN, but as this type of test is laborious, slow and time-consuming it was dismissed as soon as serological and molecular assays became available,

2.2.8.2 Present – Serological and Molecular assays

From 1982 onwards, monoclonal antibodies and polyclonal antisera were produced for the detection of FD phytoplasma (Caudwell et al., 1982; Schwarz et al., 1989). These antisera were also used for observing phytoplasmas by immunosorbent electron microscopy (ISEM) and fluorescent light microscopy (Lherminier et al., 1989). Successful differentiation between FD and phytoplasmas of the same taxonomic group (16SrV) using monoclonal antibodies was reported by Seddas and co-workers (1993, 1995, 1996). Once the first DNA probe was synthesized on phytoplasma genome sequences, recombinant DNA-based techniques were rapidly developed (Kirkpatrick et al., 1987). These techniques were affordable for the detection in herbaceous hosts, but were found to be inaccurate in woody plants (including grapevine), mainly because of the low concentration of the pathogen and erratic distribution in this host (Belli et al., 2010). The availability of the 16S rRNA gene sequences of AYp, FD and BN allowed for the development of universal PCR assays for the detection of all known phytoplasmas (Lim and Sears, 1989; Davis et al., 1993; Daire et al., 1993; Deng and Hiruki, 1991; Lee et al., 2004). These assays were further developed for the reliable identification of grapevine

phytoplasma sub-groups, based on restriction fragment length

polymorphism and highly sensitive nested-PCRs (Lee et al., 1994; Bianco et al., 1996). For faster and even more specific detection of grapevine

11 phytoplasmas, real-time RT-PCRs, nanobiotransducers, multiplex nested-PCRs, ligase detection reactions and DNA microarrays were successfully developed (Angelini et al., 2007; Firrao et al., 2005; Clair et al., 2003; Christensen et al., 2004; Frosini et al., 2002) and are currently being used by the industry to accurately detect phytoplasma species.

2.2.9 Seasonal and spatial distribution

Detecting phytoplasmas goes hand-in-hand with the distribution of the pathogen throughout a host plant. Seasonal distribution plays a big role in detecting phytoplasmas. Terlizzi and Credi (2007) reported that the proportion of BN presence was highest in summer throughout five different cultivars of grapevine, located in Italy. In winter, the number of infected grapevines clearly decreased. This seasonal distribution was also described in grapevine infected with AGYp, where detection was most reliable during summer and decreased in autumn (Constable et al., 2003). These results suggest that phytoplasmas are unevenly distributed, seldom spreading systemically through grapevines and rarely infecting them persistently from year to year (Terlizzi and Credi, 2007; Constable et al., 2003; Gibb et al., 1999; Hollingsworth et al., 2008; Seemüller et al., 1994). In Catharanthus roseus (C. roseus) plants, the colonization pattern and distribution of two „Candidatus P. asteris‟ subspecies, namely severe AYp and dwarf AYp, were generally similar over a 10 week period (Kuske and Kirkpatrick, 1992). Phytoplasmas are also known to accumulate disproportionately in Euphorbia pulcherrima source leaves, and to a lesser extent in the petioles of source leaves, whereas the accumulation of phytoplasmas is lowest in sink organs (Christensen et al., 2004). The infection level of phytoplasmas also differs greatly between host plants. Stolbur phytoplasma showed significant differences in the level of phytoplasma infection between V. vinifera cvs „Cabernet Sauvignon‟ and „Sauvignon blanc‟ (Orenstein et al., 2001). Christensen et al. (2004) reported that phytoplasma titres observed in C. roseus are significantly higher to pathogen titres seen in E. pulcherrima. Despite the long history of research on AYp, no data are available on the spatial pattern of AYp-infected plants and the change in pattern over time as disease incidence increases.

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2.2.10 Control strategies

According to Carstens (2008), no control strategy exists to cure a plant infected with phytoplasmas. The Department of Agriculture, Forestry and Fisheries in the Republic of South Africa has thus set aside multiple practices to aid in the prevention of further spread of „Ca. P. asteris‟. These include weed control and intercropping, chemical control, vineyard sanitation, propagation of material and the marking of infected grapevine in all vineyards. Techniques that are currently being investigated to aid in the control of phytoplasmas are described below.

2.2.10.1 Auxin-induced recovery

In 1968, Davies and his colleagues showed that tetracycline has a bacteriostatic effect on phytoplasmas. Unfortunately, once the treated plants were transferred to antibiotic-free medium, phytoplasma symptoms reappeared. Other substances have been shown to alter phytoplasma ultrastructure. These include β-amino-butyric acid (BABA), polyamines, putrescine, spermidine and spermine (Musetti et al., 1999). Ćurković Perica (2008) discovered that phytoplasma-infected shoots recover better on medium containing auxins, rather than benzyl-aminopurine. This technique is however dependent on the phytoplasma species. For example, „Ca. P. asteris‟ and „Ca. P. pruni‟ were susceptible to the supplementation of endogenous auxins, whereas „Ca. P. ulmi‟ and „Ca. P. solani‟ were not. Phytoplasma-infected C. roseus shoots treated with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) led to the remission of symptoms in in vitro grown plants, but did not lead to the elimination of „Candidatus P. asteris‟ (Ćurković Perica et al., 2007; Ćurković Perica, 2008). „Candidatus P. ulmi‟ infected C. roseus plants were always symptomatic when grown on medium containing 6-benzylaminopurine (BA) compared to infected shoots grown on IBA, which showed recovery (Leljak-Levanić et al., 2010). Despite the recovery of symptoms, these shoots were still found to be infected by the pathogen through the amplification of its 16S rDNA. These results show that the recovery as a remission of symptoms may or may not involve elimination of the pathogen from the host plant.

2.2.10.2 Natural recovery

The natural remission of symptoms has been observed in several grapevine cultivars worldwide. Recovery was first observed in France and Italy in FD

13 infected vines, followed by recovery of BN in grapevine (Caudwell, 1961; Belli et al., 1978; Osler et al., 1993). This phenomenon has recently been described in apples infected with apple proliferation phytoplasma and apricots infected with European stone fruit yellows (Musetti et al., 2004). In naturally recovered vines, remission of symptoms is often accompanied by the disappearance of the infection (Osler et al., 2006; Zorloni et al., 2008). Osler and colleagues (1999) suggested that systemic acquired resistance (SAR) might be involved in apple and pear recovery. Recently, an increase of reactive oxygen species (ROS) has been detected in grapevine displaying FD recovery (Musetti et al., 2007). So far, the information available is still insufficient for a clear explanation of recovery, although it seems reasonable that interactions between the pathogen, the host and the environment may play a key role, as well as the involvement of grapevine bacterial or fungal endophytes (Belli et al., 2010; Musetti et al., 2007; Bulgari et al., 2009; Martini et al., 2009).

2.2.10.3 Hot water treatment

Another control strategy to cure dormant woody plant material from phytoplasmas is the use of heat or hot water treatment. Tassart-Subirats et al. (2003) used hot water treatment to eliminate FD from grapevine sections. As hot water treatment may interfere with the vitality of woody propagated material, it must be carefully applied under the correct temperature/time regimes and with the proper equipment (Mannini, 2007).

2.2.10.4 Abiotic stresses

Recovery of phytoplasma infections can also be promoted by exposing the grapevine to abiotic stress, such as uprooting followed by immediate transplanting, partial uprooting or pulling and pruning and pollarding (Osler et al., 1993; Romanazzi and Murolo, 2008; Borgo and Angelini, 2002). Partial uprooting has been effective in inducing recovery in almost all grapevine cvs „Chardonnay‟, „Verdicchio‟ and „Sangiovese‟ grafted onto Kober 5BB (Romanazzi and Murolo, 2008). After the first year of recovery from BN obtained by partial uprooting, V. vinifera cv „Primitivo‟ had a similar trend in photosynthesis and respiration compared to healthy plants (Murolo et al., 2009).

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2.2.10.5 Expression of antimicrobial peptides in transgenic plants

In recent years, genetic modification has become an option for inducing disease resistance in plants. Du and his colleagues (2005) reported an increase in plant resistance against witches‟ broom disease in greenhouse transgenic Paulownia plants, expressing the antimicrobial peptide Shiva-1. Transgenic tobacco plants expressing a scFv antibody specific for the immunodominant membrane protein of Stolbur phytoplasma showed no significant resistance when the phytoplasma was transmitted to the plants by grafting or by its vector (Le Gall et al., 1998; Malembic-Maher et al., 2005). For engineering genetic resistance to phytoplasmas in grapevine, it could be more beneficial to engineer resistance in rootstocks, rather than individual grapevine cultivars. As phytoplasmas are known to move to the roots during winter, confronting them at this time with resistant rootstock could greatly decrease the chance of recurrence in the following year (Constable et al., 2003). As the knowledge on plant genes inducing phytoplasma resistance is still very scarce, opportunities to select resistant varieties by traditional or molecular assisted breeding is limited (Belli et al., 2010). Keeping the public‟s acceptance and environmental safety issues of genetically modified plants in mind, transgenic strategies for creating resistance of grapevine towards pathogens, remains challenging. Open and proactive dialogues between the scientific community and the public should be greatly encouraged, as they shed light on the benefits and practical usefulness of this technology.

2.3 Antimicrobial peptides

Grapevines are exposed to many plant pathogens and the resulting diseases may cause major economic losses. Chemical pesticides are being used to combat this global problem. However, pesticide usage has proven to be harmful to the environment and consumers health, and an overuse may lead to pathogen resistance (Keymanesh and Sotani, 2009). Scientists have therefore started looking at elements that present sustainable resistance to a broad range of pests and pathogens and that are safe for the host organism with no side effect on the environment.

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2.3.1 General Information

Antimicrobial peptides (AMPs) form part of the innate, non-specific immune system shared by plants, humans and animals and are safe for the host organism with no side effects on the environment (Brown and Hancock, 2006). Rydlo et al., (2006) reported that organisms produce AMPs in response to microbial infection, or they produce the peptides constitutively and store them in large quantities for later use. Antimicrobial peptides are made up of 12-50 amino acid residues and have shown to be effective against Gram-negative and Gram-positive bacteria, fungi, viruses and eukaryotic parasites (Wang et al., 2006). Generally these peptides are cationic, rich in cysteine and amphipatic, giving them a great affinity for the pathogens membrane. Antimicrobial peptides are grouped into two groups based on their electrostatic charge. The positively charged peptides are divided into β-sheets, α-helices, extended helices and loop structures (Powers and Hancock, 2003). The second electrostatic group, namely the non-cationic peptides, are grouped into anionic and aromatic peptides and are very scarce. According to Keymanesh and Soltani (2009), some AMPs are produced solely by bacteria and are termed non-ribosomally synthesized peptides, whilst the ribosomally synthesized peptides are made by all organisms. Most peptides are not used in their native form to confer resistance to pathogens due to factors influencing the AMP activity, such as an increase in potency of anti-pathogen activity, reduction of their haemolytic effect or inhibition by host proteases. Scientists are therefore using analogue peptides or derivatives of the original AMPs (Lee et al., 2002). Synthetic peptides are obtained by solid-phase methods and procedures using combinatorial chemistry (Andreu et al., 1983; Monroc et al., 2006). D4E1, a synthetic analogue of the cecropin family is more stable and potent than its native counterpart, and shows minimal cytotoxic activities against mammalian cells. This synthetic peptide demonstrates inhibition of spore germination of various fungal pathogens and also affects bacterial pathogens (Jacobi et al., 2000; Rajaekaran et al., 2009).

2.3.2 Plant AMPs

Plants have two broad mechanisms of pathogen resistance. Firstly, they may use the structures and compounds synthesized throughout their development to confer resistance against pathogens (constitutive resistant factors), or they make use of the induction mechanism which is activated after contact with the pathogen (induced

16 resistant factors) (Castro and Fontes, 2005). Both of these mechanisms involve the expression of peptides which present direct antimicrobial activity. Plant AMPs are grouped into different families based on their sequence similarity and activity towards certain pathogens. These families include the cyclotides, thionins (now named defensins), snakins, 2S albumins, hevein-type proteins and lipid transfer proteins, among many others (Peligrini et al., 2011). The first plant defensin isolated from Vitis vinifera is Vv-AMP1 and was characterized by de Beer and Vivier (2008). Vv-AMP1 shows a strict tissue-specific and developmentally regulated expression pattern and is strongly antifungal. In 2008, de Beer and Vivier proved that Vv-AMP1 showed a very high level of activity against the pathogens Fusarium oxysporum and Verticillium dahlia in grapevine.

2.3.3 Mechanisms of cell death induced by AMPs

During pathogen infection, the pathogen will utilize substances from the plant host to facilitate its movement through the physical barriers presented by the plant (Castro and Fontes, 2005). The pathogen will also obtain nutrients from the plant for its own survival, while secreting multiple substances into the host which degrade the cell wall, interrupt metabolic functions or pathways, promote imbalance in the plants hormonal system and block the water translocation mechanism throughout the vascular system (Castro and Fontes, 2005).

Once the plant has come into contact with the pathogen, a series of peptides are expressed with some of them showing antimicrobial properties. The cationic peptides are attracted electrostatically to negatively charged molecules found in the pathogen membrane, but they may also interact with membrane lipids by specific receptors at the surface (Sitaram and Nagaraj, 1999). Generally, once the peptide threshold concentration is reached, AMPs accumulate on the membrane surface to direct inner components for cell lyses through pore formation. Three processes of pore formation have been summarized by Pelegrini and colleagues (2011). The barrel-stave mechanism consists of peptide aggregates forming a barrel-ring around an aqueous pore (Figure 3A). Once the peptides have bound to the membrane phospholipids and the threshold concentration has been reached, they start forming a barrel-ring to open a pore. The core of the barrel is made up of the hydrophilic portions, whereas the hydrophobic portion interacts with the membrane phospholipids. The second process of pore formation, namely the toroidal pore, is

17 very similar to the barrel-stave mechanism. The shape of the pore is similar; however the pore is composed of overlapping peptides and membrane lipids. The last mode of pore formation is the carpet mechanism (Figure 3B) (Pelegrini et al., 2011). Initially the peptides bind to the pathogen membrane electrostatically giving the appearance of a carpet on the bacterial membrane surface. This causes phospholipid displacement that alters the membrane fluidity and reduces barrier properties of the membrane.

Figure 3: The processes of pore formations by AMPs. A: The barrel-steve mechanism. B: The

carpet mechanism (Pelegrini et al., 2011)

Once bound to the pathogen‟s membrane, AMPs can activate several pathways that will lead to cell death (Figure 4). Some peptides, as mentioned before will form pores. Ions and energy gradients dissipate through these pores and cause cell lysis within minutes (Figure 4A) (Bowman et al., 2003). On the other hand, some peptides do not disrupt the pathogen membrane. Instead, bacteria exposed to these peptides show a decrease in protein synthesis, indicating that the peptide crosses the cell membrane to interact with intracellular targets and inhibit nucleic acid or protein synthesis, leading to cell death. (Figure 4B).